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CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.

Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R - J. Cell Biol. (2001)

Bottom Line: The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans.This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells.These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

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HECA-452–reactive CD44 functions as an E-selectin ligand. (A) KG1a membrane protein (10 μg) was resolved on a reducing 6% SDS-PAGE gel, blotted onto PVDF membrane, stained with HECA-452, and HECA-452 immunoblots blots were rendered transparent with 10% glycerol. CHO-E cells (2 × 106/ml) were then perfused over these blots at a defined shear stress of 3.8 dynes/cm2. Several HECA-452–stained bands from KG1a membrane protein–supported, E-selectin–dependent CHO-E cell rolling. (B) KG1a membrane proteins (10 μg) were treated with N-glycosidase F, separated on a reducing 6% SDS-PAGE gel, and immunostained with HECA-452. (C) Immunoprecipitated PSGL-1 was resolved on a reducing 6% SDS-PAGE gel and Western blotted with either HECA-452 (left) or anti–PSGL-1 antibody 4H10 (right). (lane 1) 10 μg of total KG1a membrane protein; (lane 2) immunoprecipitated PSGL-1 from 100 μg of KG1a membrane protein; (lane 3) 100 μg of total KG1a membrane protein, and (lane 4) immunoprecipitated PSGL-1, from 100 μg of KG1a membrane protein. Note that HECA-452–stained bands at 140 and 220 kD correspond to PSGL-1. (D) Isotype control or Hermes-1 immunoprecipitated CD44 from KG1a membrane protein (50 μg) was resolved on a reducing 9% SDS-PAGE gel and immunoblotted with HECA-452. Immunoprecipitated CD44 from KG1a membrane proteins (50 μg) treated with N-glycosidase F was also immunoblotted with HECA-452. Though CHO-E cell rolling frequencies are presented as the mean ± SD of E-selectin–mediated cell rolling at 3.8 dynes/cm2 measured on the 100-kD isoform of CD44, no CHO-E rolling was observed along the entire length of the N-glycosidase F–treated lane.
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Figure 3: HECA-452–reactive CD44 functions as an E-selectin ligand. (A) KG1a membrane protein (10 μg) was resolved on a reducing 6% SDS-PAGE gel, blotted onto PVDF membrane, stained with HECA-452, and HECA-452 immunoblots blots were rendered transparent with 10% glycerol. CHO-E cells (2 × 106/ml) were then perfused over these blots at a defined shear stress of 3.8 dynes/cm2. Several HECA-452–stained bands from KG1a membrane protein–supported, E-selectin–dependent CHO-E cell rolling. (B) KG1a membrane proteins (10 μg) were treated with N-glycosidase F, separated on a reducing 6% SDS-PAGE gel, and immunostained with HECA-452. (C) Immunoprecipitated PSGL-1 was resolved on a reducing 6% SDS-PAGE gel and Western blotted with either HECA-452 (left) or anti–PSGL-1 antibody 4H10 (right). (lane 1) 10 μg of total KG1a membrane protein; (lane 2) immunoprecipitated PSGL-1 from 100 μg of KG1a membrane protein; (lane 3) 100 μg of total KG1a membrane protein, and (lane 4) immunoprecipitated PSGL-1, from 100 μg of KG1a membrane protein. Note that HECA-452–stained bands at 140 and 220 kD correspond to PSGL-1. (D) Isotype control or Hermes-1 immunoprecipitated CD44 from KG1a membrane protein (50 μg) was resolved on a reducing 9% SDS-PAGE gel and immunoblotted with HECA-452. Immunoprecipitated CD44 from KG1a membrane proteins (50 μg) treated with N-glycosidase F was also immunoblotted with HECA-452. Though CHO-E cell rolling frequencies are presented as the mean ± SD of E-selectin–mediated cell rolling at 3.8 dynes/cm2 measured on the 100-kD isoform of CD44, no CHO-E rolling was observed along the entire length of the N-glycosidase F–treated lane.

Mentions: To examine the E-selectin ligand activity of all HECA-452–reactive KG1a membrane proteins, we used a new method for assessing the adhesive interactions under shear flow between selectin-expressing whole cells and proteins immobilized on Western blots (Dimitroff et al. 2000). Membrane proteins were separated on 6% SDS-PAGE gel, transferred onto PVDF membrane, and then stained with HECA-452. Component membrane proteins were analyzed for their ability to support CHO-E cell rolling under defined hydrodynamic flow conditions. We reproducibly observed E-selectin ligand activity on HECA-452–stained bands at 100, 120, 140, 190, and 220 kD, but not at 74 kD (Fig. 3 A). CHO-mock cells showed no interactions with any bands. Specificity for E-selectin was demonstrated by the abrogation of CHO-E cell rolling in the presence of either 5 mM EDTA or anti–E-selectin functional blocking Abs. On HL60 cells, CHO-E cell rolling was observed only over the broad 140-kD HECA-452–immunostained band (i.e., PSGL-1/CLA) (data not shown). To determine whether E-selectin–binding determinants reside on N-glycans, we treated KG1a membrane protein with N-glycosidase F. De–N-glycosylated proteins were resolved by SDS-PAGE and analyzed for HECA-452 reactivity and E-selectin ligand activity. N-glycosidase F treatment markedly diminished HECA-452 staining (Fig. 3 B) and completely abolished CHO-E cell rolling on all proteins on the blot, indicating that all glycoprotein E-selectin binding determinants on KG1a cells are displayed exclusively on N-glycans.


CD44 is a major E-selectin ligand on human hematopoietic progenitor cells.

Dimitroff CJ, Lee JY, Rafii S, Fuhlbrigge RC, Sackstein R - J. Cell Biol. (2001)

HECA-452–reactive CD44 functions as an E-selectin ligand. (A) KG1a membrane protein (10 μg) was resolved on a reducing 6% SDS-PAGE gel, blotted onto PVDF membrane, stained with HECA-452, and HECA-452 immunoblots blots were rendered transparent with 10% glycerol. CHO-E cells (2 × 106/ml) were then perfused over these blots at a defined shear stress of 3.8 dynes/cm2. Several HECA-452–stained bands from KG1a membrane protein–supported, E-selectin–dependent CHO-E cell rolling. (B) KG1a membrane proteins (10 μg) were treated with N-glycosidase F, separated on a reducing 6% SDS-PAGE gel, and immunostained with HECA-452. (C) Immunoprecipitated PSGL-1 was resolved on a reducing 6% SDS-PAGE gel and Western blotted with either HECA-452 (left) or anti–PSGL-1 antibody 4H10 (right). (lane 1) 10 μg of total KG1a membrane protein; (lane 2) immunoprecipitated PSGL-1 from 100 μg of KG1a membrane protein; (lane 3) 100 μg of total KG1a membrane protein, and (lane 4) immunoprecipitated PSGL-1, from 100 μg of KG1a membrane protein. Note that HECA-452–stained bands at 140 and 220 kD correspond to PSGL-1. (D) Isotype control or Hermes-1 immunoprecipitated CD44 from KG1a membrane protein (50 μg) was resolved on a reducing 9% SDS-PAGE gel and immunoblotted with HECA-452. Immunoprecipitated CD44 from KG1a membrane proteins (50 μg) treated with N-glycosidase F was also immunoblotted with HECA-452. Though CHO-E cell rolling frequencies are presented as the mean ± SD of E-selectin–mediated cell rolling at 3.8 dynes/cm2 measured on the 100-kD isoform of CD44, no CHO-E rolling was observed along the entire length of the N-glycosidase F–treated lane.
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Related In: Results  -  Collection

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Figure 3: HECA-452–reactive CD44 functions as an E-selectin ligand. (A) KG1a membrane protein (10 μg) was resolved on a reducing 6% SDS-PAGE gel, blotted onto PVDF membrane, stained with HECA-452, and HECA-452 immunoblots blots were rendered transparent with 10% glycerol. CHO-E cells (2 × 106/ml) were then perfused over these blots at a defined shear stress of 3.8 dynes/cm2. Several HECA-452–stained bands from KG1a membrane protein–supported, E-selectin–dependent CHO-E cell rolling. (B) KG1a membrane proteins (10 μg) were treated with N-glycosidase F, separated on a reducing 6% SDS-PAGE gel, and immunostained with HECA-452. (C) Immunoprecipitated PSGL-1 was resolved on a reducing 6% SDS-PAGE gel and Western blotted with either HECA-452 (left) or anti–PSGL-1 antibody 4H10 (right). (lane 1) 10 μg of total KG1a membrane protein; (lane 2) immunoprecipitated PSGL-1 from 100 μg of KG1a membrane protein; (lane 3) 100 μg of total KG1a membrane protein, and (lane 4) immunoprecipitated PSGL-1, from 100 μg of KG1a membrane protein. Note that HECA-452–stained bands at 140 and 220 kD correspond to PSGL-1. (D) Isotype control or Hermes-1 immunoprecipitated CD44 from KG1a membrane protein (50 μg) was resolved on a reducing 9% SDS-PAGE gel and immunoblotted with HECA-452. Immunoprecipitated CD44 from KG1a membrane proteins (50 μg) treated with N-glycosidase F was also immunoblotted with HECA-452. Though CHO-E cell rolling frequencies are presented as the mean ± SD of E-selectin–mediated cell rolling at 3.8 dynes/cm2 measured on the 100-kD isoform of CD44, no CHO-E rolling was observed along the entire length of the N-glycosidase F–treated lane.
Mentions: To examine the E-selectin ligand activity of all HECA-452–reactive KG1a membrane proteins, we used a new method for assessing the adhesive interactions under shear flow between selectin-expressing whole cells and proteins immobilized on Western blots (Dimitroff et al. 2000). Membrane proteins were separated on 6% SDS-PAGE gel, transferred onto PVDF membrane, and then stained with HECA-452. Component membrane proteins were analyzed for their ability to support CHO-E cell rolling under defined hydrodynamic flow conditions. We reproducibly observed E-selectin ligand activity on HECA-452–stained bands at 100, 120, 140, 190, and 220 kD, but not at 74 kD (Fig. 3 A). CHO-mock cells showed no interactions with any bands. Specificity for E-selectin was demonstrated by the abrogation of CHO-E cell rolling in the presence of either 5 mM EDTA or anti–E-selectin functional blocking Abs. On HL60 cells, CHO-E cell rolling was observed only over the broad 140-kD HECA-452–immunostained band (i.e., PSGL-1/CLA) (data not shown). To determine whether E-selectin–binding determinants reside on N-glycans, we treated KG1a membrane protein with N-glycosidase F. De–N-glycosylated proteins were resolved by SDS-PAGE and analyzed for HECA-452 reactivity and E-selectin ligand activity. N-glycosidase F treatment markedly diminished HECA-452 staining (Fig. 3 B) and completely abolished CHO-E cell rolling on all proteins on the blot, indicating that all glycoprotein E-selectin binding determinants on KG1a cells are displayed exclusively on N-glycans.

Bottom Line: The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans.This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells.These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.

ABSTRACT
E-selectin plays a critical role in mediating tissue-specific homing of T cells into skin, and of primitive hematopoietic progenitor cells (HPCs) into bone marrow (BM). Though it is known that a glycoform of PSGL-1 (CLA) functions as the principal E-selectin ligand on human T lymphocytes, the E-selectin ligand(s) of human HPCs has not been identified. We used a shear-based adherence assay to analyze and define the E-selectin ligand activity of membrane proteins from human HPCs. Our data show that PSGL-1 expressed on human HPCs is an E-selectin ligand, and that HPCs also express a previously unrecognized E-selectin ligand, CD44. The E-selectin ligand activity of CD44 is conferred by the elaboration of sialylated, fucosylated binding determinants on N-glycans. This glycoform of CD44 is expressed on primitive CD34+ human HPCs, but not on more mature hematopoietic cells. Under physiologic flow conditions, this molecule mediates E-selectin-dependent rolling interactions over a wider shear range than that of PSGL-1, and promotes human HPC rolling interactions on E-selectin expressed on human BM endothelial cells. These findings offer new insights into the structural biology and physiology of CD44, and into the molecular basis of E-selectin-dependent adhesive interactions that direct homing of human HPC to BM.

Show MeSH
Related in: MedlinePlus